Extension is achieved by using the loosened nucleotides of each base to grow the complementary DNA strand. The end result is two double-stranded products of DNA.
The temperature that is used during the extension phase is dependent on the DNA polymerase that is used. Generally, at this stage, the reaction mixture is heated to a temperature intermediate between the denaturation and annealing temperatures. Because the reaction mixture is heated, most PCR techniques employ a heat-stable DNA polymerase.
One of the most widely used heat-stable DNA polymerases is the
Taq polymerase which is an enzyme originally isolated from the bacterium
Thermus aquaticus. Taq polymerase is involved in extending the DNA primers in the 5' to 3' direction. The optimal operating temperature for Taq polymerase is 72°C. The extension time depends upon the length of the target DNA sequence but a general rule of thumb is one thousand bases per minute at optimal temperature. Under ideal conditions, the amount of target DNA should double in the extension step.
